Background

PM100117 and PM100118 are glycosylated polyketides with remarkable
antitumor activity, which derive from the marine symbiotic actinobacteria
Streptomyces caniferus GUA-06-05-006A.
Structurally, PM100117 and PM100118 are composed of a macrocyclic lactone, three
deoxysugar units and a naphthoquinone (NQ) chromophore that shows a clear
structural similarity to menaquinone.

Results

Whole-genome sequencing of S.
caniferus GUA-06-05-006A has enabled the identification of
PM100117 and PM100118 biosynthesis gene cluster, which has been characterized on
the basis of bioinformatics and genetic engineering data. The product of four
genes shows high identity to proteins involved in the biosynthesis of
menaquinone via futalosine. Deletion of one of these genes led to a decay in
PM100117 and PM100118 production, and to the accumulation of several derivatives
lacking NQ. Likewise, five additional genes have been genetically characterized
to be involved in the biosynthesis of this moiety. Moreover, the generation of a
mutant in a gene coding for a putative cytochrome P450 has led to the production
of PM100117 and PM100118 structural analogues showing an enhanced in vitro
cytotoxic activity relative to the parental products.

Conclusions

Although a number of compounds structurally related to PM100117 and
PM100118 has been discovered, this is, to our knowledge, the first insight
reported into their biosynthesis. The structural resemblance of the NQ moiety to
menaquinone, and the presence in the cluster of four putative menaquinone
biosynthetic genes, suggests a connection between the biosynthesis pathways of
both compounds. The availability of the PM100117 and PM100118 biosynthetic gene
cluster will surely pave a way to the combinatorial engineering of more
derivatives.

Actinobacteria is an extensive phyla within the domain bacteria with a
wide distribution in nature, encompassing both terrestrial and aquatic environments
[1, 2]. Numerous actinobacteria species have an outstanding medical
value as producers of cancer chemotherapeutic drugs [3], among other biologically active compounds [4, 5]. Terrestrial actinobacteria are the source of the vast majority of
natural antitumor agents discovered up to date, many of which are the base of
currently available chemotherapeutic treatments or are in advance clinical trials
[6]. However, in spite of the
enormous potential of soil actinobacteria as producers of antitumor agents, the rate
of discovery of new antitumor drugs and other bioactive compounds from the
terrestrial environment has nowadays decayed [7–9]. This, together
with the worldwide-rising occurrence of cancer and the appearance of multi-drug
resistant tumor cell lines, urges to extend the screening of new and improved
chemotherapeutic products to less explored environments.

Ocean is a major component of the biosphere and an example of
unexploited habitat with the potential to host the chemical and biological diversity
required for the discovery of novel anticancer agents. A staggering diversity of
actinobacteria species has been isolated from different marine substrates such as
sediments seawater, seaweeds or mangroves [2]. Furthermore, tissues of mollusks and invertebrates are the
niche of remarkably diverse symbiotic actinobacteria populations many of which could
have lost the ability to grow independently of their host [10]. Some of the symbiotic functions
attributable to symbiotic actinobacteria entail the production of bioactive
secondary metabolites (e.g., for host defense) with potential pharmaceutical
significance. Indeed, many natural products isolated from diverse marine
invertebrates have been proven to be produced by their symbiotic microorganisms.
That is the case, for example, of the dibenzodiazepinone diazepinomicin
[11], the thiodepsipeptide
thiocoraline [12] and the polyketide
bryostatin [13], three potent antitumor
drug leads; or the tetrahydroisoquinoline ecteinascidin 743 [14], an anticancer drug in current clinical
use.

Although the screening of antitumor natural products from yet
unexploited environments is a valid plan to alleviate the problem of the apparent
chemical exhaustion of terrestrial sources, the use of metabolic engineering and
combinatorial biosynthesis strategies intended to generate novel analogues from
known natural agents [15–17], can be a no less interesting approach.
However, application of these genetic engineering strategies requires to a certain
extent detailed knowledge on the genetic and biochemical basis of the biosynthesis
of relevant natural metabolites. Hence, the identification and characterization of
biosynthetic gene clusters is not only an invaluable tool for the elucidation of the
biosynthesis pathway of bioactive natural agents, but also an essential requirement
for tackling the combinatorial engineering of novel analogues. In parallel with the
development of these genetic engineering approaches, advances in next-generation
whole-genome sequencing techniques [18]
has favored the availability of a growing number of genomes from actinobacteria
producing clinical attractive bioactive compounds. Mining of these genomes has
enabled the identification and analysis of an exponentially increasing number of
clusters [19], setting the stage for
subsequent combinatorial engineering of novel derivatives. Furthermore, genome
analysis by enhanced bioinformatics platforms associated to database [20–24] allows for disclosing the chemical diversity potentially
hosted in theses actinobacteria, contributing to widen the availability of natural
products by activation of cryptic clusters [19].

In a recent publication [25], the discovery of PM100117 and PM100118 was reported. These
glycosylated polyketide compounds, with remarkable antitumor activity, are produced
by Streptomyces caniferus GUA-06-05-006A, a
symbiotic actinobacteria isolated from the marine sponge Filograna sp. The structures of PM100117 and PM100118, consisting of
a macrocyclic lactone, three deoxysugars and a 1, 4-naphthoquinone chromophore
(Fig. 1), share a clear similarity with
other antitumor polyketide compounds, including langkolide [26] and the promising anticancer drug lead GT35
[27]. Given their attractive as
potential anticancer clinical drugs, PM100117 and PM100118 represent interesting
targets for the combinatorial engineering of novel derivatives. The goals of this
work were the identification and characterization of the PM100117 and PM100118
biosynthesis gene cluster, and engineering the gene cluster to generate novel
derivatives with improved biological activity.

The first step to accomplish the identification of the PM100117 and
PM100118 biosynthesis gene cluster was sequencing the S.
caniferus GUA-06-05-006A chromosome. Whole-genome sequencing of
this strain generated a total number of 548,579 paired-end reads with an average
length of 372.5 nucleotides, producing a total sequence of 204.3 Mb. This
represents a 20-fold coverage of the chromosome sequence, which is estimated in
9.8 Mb. De novo assembly of those sequences resulted in 907 contigs. Median
(N50) of the contig assembly was 20.9 Kb, and the largest was around 119.3 Kb.
Subsequent contig arrangement rinsed 33 scaffolds (mean: 276.2 Kb; N50: 1.5 Mb)
generating a draft genome of 9.1 Mb with a G + C content of 70.63 %. In silico genome analyses with the antibiotics and
Secondary Metabolite Analysis Shell (antiSMASH) algorithm [22] revealed the presence of 8582 open
reading frames (ORFs), having at least 5045 proteins an assigned putative
function. Sequence analyses with antiSMASH also showed the presence of 32 gene
clusters potentially involved in the biosynthesis of secondary metabolites.
Seven of these clusters were identified as containing gene sequences belonging
to the type I (four clusters), II (two clusters) and III (one cluster) family of
polyketide synthases (PKS). Likewise, genome sequence analysis detected nine
additional gene clusters comprising modular enzyme-coding genes such as
non-ribosomal peptide synthetase (NRPS, seven clusters) and hybrid PKSI-NRPS
genes (two clusters). Other products from these gene clusters include one
nucleoside, five terpenes and three butyrolactones, as well as compounds with a
peptidic backbone such as three siderophores, three lantipeptides and one
ectoine.

Identification of PM100117 and PM100118 biosynthetic gene cluster

PM100117/18 chemical structures have been previously elucidated by
nuclear magnetic resonance (NMR) spectroscopy [25]. They contain a 48 carbons aglycone that is likely
biosynthesized by the condensation of 21 ketide moieties. The aglycone forms a
36-membered macrolactone ring and is decorated with a side chain consisting of
three 2,6-dideoxy sugars and a 1,4-naphtoquinone chromophore (Fig. 1). The first sugar moiety attached to the
aglycone is l-axenose, which can be linked
either to l-2-deoxy-fucose (PM100117) or
l-rhodinose (PM100118) as second
sugar. The second sugar unit is linked to the naphtoquinone moiety, which shares
a clear structural similarity with menaquinone (MK). An l-rhodinose moiety attached to the naphtoquinone
structure represents the third deoxysugar of the PM100117/18 glycosylation
pattern. Based on these structural features, glycosyltransferase and sugar
biosynthetic genes, as well as a minimum of 21 PKS modules, are presumably
involved in the biosynthesis of PM100117/18. Among the seven PKS gene clusters
identified by antiSMASH, one of them contained ORFs designated with such
putative functions, representing the most suitable candidate cluster to
accomplish PM100117/18 biosynthesis. The putative PM100117/18 cluster covers a
171 kb region and contains 54 ORFs (Fig. 2a) coding for proteins with the putative functions listed in
Table 1. The involvement of this
cluster in PM100117/18 biosynthesis was demonstrated by inactivation of the PKS
gene gonP1. The analysis of the resulting
mutant strain showed that gonP1 inactivation
abolished PM100117/18 biosynthesis, thus confirming the implication of this
cluster in the production of PM100117/18 (Fig. 2b).

Fig. 2

Identification and organization of the PM100117 and
PM100118 biosynthetic gene cluster. a Organization of the PM100117 and PM100118 gene
cluster. The proposed gene functions are listed in
Table 1. b UPLC analysis of PM100117
(1) and PM100118
(2) production in
Streptomyces caniferus
GUA-06-05-006A wild type (GUA) and mutant gonP1−

In silico analysis of PM100117 and PM100118
gene cluster and proposed biosynthesis pathway

Polyketide ring and post-PKS modifications

The PM100117/18 cluster encompasses seven contiguous PKS genes
(gonP1–gonP7) coding for a multimodular PKS that comprises a
loading domain (LD) and 20 extension modules (M1–M20), in agreement with the
21 condensation steps required for the biosynthesis of the PM100117/18
macrolide ring. Sequence analysis of GonP1–GonP7 allowed to define
ketosynthase (KS or KSQ), acyltranferase (AT), ketoreductase (KR),
dehydratase (DH), enoylreductase (ER) and acyl-carrier-protein (ACP)
domains. LD contains a ketosynthase (KSQ) domain, in which the essential
cysteine of the conserved DTxCSxS sequence at the active site is replaced by
glutamine [28]. Sequence
alignment of the cluster PKS domains active sites is shown as supplementary
data (Additional file 1: Figure
S1). AntiSmash analysis also predicted substrate specificity of AT domains
within the modular PKS for methylmalonyl-CoA (ATp) in seven modules (LD,
M1–M4, M14 and M19) and for malonyl-CoA (ATa) in 14 modules (M5–M13, M15–M18
and M20). The organization of the PKS genes and the proposed biosynthesis
pathway of the PM100117/18 macrolide backbone are shown in Fig. 3. Overall, the predicted substrate
specificity of AT domains alongside the disposal of the defined KR, DH and
ER domains are consistent with the hypothetical elongation through collinear
reactions of the polyketide chain. Only a few discrepancies are found
between this biosynthesis model and the chemical structure of the polyketide
moiety, suggesting the inactivity/inability of certain PM100117/18 PKS
domains or the occurrence of post-PKS modifications. The presence of a DH
domain in M6 (GonP3), as well as a DH and an ER domain in M9 (GonP4),
apparently disturbs the collinear biosynthesis of the polyketide, given the
presence of hydroxyl groups at carbon (C) 31 and C25 of the polyketide
skeleton (Fig. 3). Nonetheless, a
detailed sequence analysis of the M6 DH domain revealed an arginine in place
of an otherwise conserved histidine in the NADPH motif LxxHxxxGxxxxP
[29] at the active site
(Aditional file 1: Figure S1).
This, together with a shorter C-terminal sequence in comparison with other
DH domains within the cluster PKSs, suggests that M6 DH is most possibly
inactive. By contrast, sequence analysis did not show any alteration of the
M9 DH and ER domains, which are probably functional. Thus, the unexpected
presence of these domains could be explained by a “domain skipping”
mechanism preventing dehydrogenation of C25 during polyketide elongation
[30]. Alternatively, the
presence of a hydroxyl group at C25 can be the result of a post-PKS
modification following polyketide biosynthesis, catalyzed by any of the two
putative oxygenases coded by gonCP and
orf9. Moreover, the involvement of
the M13 DH domain (GonP5) in the polyketide biosynthesis is unlikely due to
the lack of a KR domain in this module catalyzing a previous ketoreduction
reaction. One last inconsistency is found between the substrate specificity
of the AT domain of M19 (GonP7), which is predicted to utilize
methylmalonyl-CoA as extension unit, and the absence of a methyl group at C4
(Fig. 3). However, the presence
in this domain of a YASH motif, which specifies utilization of
methylmalonyl-CoA [31], can be
confirmed as shown in the alignment of cluster AT active sites (Additional
file 1: Figure S1). Despite this,
no PM100117/18 analogues containing an additional methyl group at C4 have
been detected in S. caniferus
GUA-06-05-006A cultures.

Sugars biosynthesis

A distinctive feature of the PM100117/18 biosynthesis gene
cluster, among the 32 clusters for secondary metabolites hosted by S. caniferus GUA-06-05-006A, is the presence of
ORFs with assigned putative functions implicated in biosynthesis or transfer
of deoxysugars. Based on these putative functions the biosynthesis pathway
of l-axenose, l-2-deoxi-fucose and l-rhodinose can be predicted
(Fig. 4). Proteins GonGS and
GonD2, putative NDP-glucose synthase and NDP-glucose 4,6-dehydratase,
respectively, might catalyze the biosynthesis of the key intermediate
NDP-4-keto-6-deoxy-d-glucose
[32], which should be then
transformed into NDP-4-keto-2,6-dideoxy-d-glucose (2,6-DG) by the activity of the putative
NDP-hexose 2,3-dehydratase GonD1 and the putative NDP-hexose 3-ketoreductase
GonR3. Biosynthesis of l-2-deoxi-fucose from 2,6-DG requires 3,5-epimerization
(3,5-EPI) and C4-ketoreduction (C4-KR) reaction steps, which are possibly
catalyzed by the putative dTDP-deoxyglucose 3,5-epimerase GonE and one of
the putative NDP-4-keto-6-deoxyhexose reductases GonR1 or GonR2,
respectively. In addition to 3,5-EPI and C4-KR reactions, biosynthesis of
l-axenose from 2,6-DG involves a
C3-metylation step, presumably catalyzed by the putative
NDP-hexose-3-C-methyltransferase GonCM. Moreover, biosynthesis of l-rhodinose requires the C3-dehydration
reaction of 2,6-DG, possibly catalyzed by the putative
NDP-hexose-3,4-dehydratase enzyme GonD3, followed by 3,5-EPI and C4-KR.
After macrolactone formation, four putative glycosyltransferase-coding genes
(gonG1, gonG2, gonG3 and
gonG4) could be involved in the
transfer of the three deoxysugar moieties to generate PM100117/18.

Fig. 4

Schematic representation of the proposed
biosynthesis pathway of PM100117 and PM100118 deoxysugar
moieties

Naphthoquinone (NQ) biosynthesis

A further peculiarity of the PM100117/18 biosynthesis cluster
is the presence of four ORFs (gonM1,
gonM2, gonM3 and gonM4) with
high identity to genes previously reported as involved in menaquinone
biosynthesis via futalosine. This newly discovered MK pathway was first
described in S. coelicolorA3(2)
[33–35] but bioinformatics analyses suggest
its presence also in other bacteria lacking the classic MK biosynthetic
pathway via isochorismate [33,
36, 37]. Sequence analysis reveals a high
degree of identity (I) and similarity (S) of GonM1, GonM2 and GonM4 to the
S. coelicolor A3(2) proteins
SCO4506/MqnA (I: 60.1 %, S: 74.4 %), SCO4550/MqnC (I: 85 %, S: 91.6 %) and
SCO4326/MqnD (I: 69.5 %, S: 77.1 %), respectively. These S. coelicolor A3(2) proteins, together with
SCO4327/MqnB, have been previously shown to be involved in the biosynthesis
of the futalosine MK pathway intermediate 1,4-dihydroxy 6-napthoic acid
(DH6N) [33–35]. Furthermore, GonM3 shares sequence
resemblance with SCO4494/MqnE (I: 93 %, S: 96.4), a protein proposed to
catalyze the biosynthesis of aminodeoxyfutalosine [38], an alternative substrate for MK
biosynthesis. An additional copy of genes coding for proteins with high
identity and similarity to S. coelicolor
A3(2) MqnA (I: 56.6 %, S: 62.2 %), MqnB (I: 68.1 %, S: 72.9 %), MqnC (I:
92.7 %, S: 97.0 %) and MqnD (I: 72.2 %, S: 75.7 %) has been detected in the
S. caniferus GUA-06-05-006A genome,
outside the PM100117/18 gene cluster. The structural similarity of DH6N with
the NQ unit led us to suspect a role of genes gonM1–gonM4 on the
biosynthesis of this compound. Other ORFs potentially involved in the
biosynthesis of NQ code for AMP-dependent synthetase and ligase GonSL,
3-oxoacyl-ACP synthase III GonS1 and GonS2, type I PKS GonP8 and
methyltransferase GonMT. GonSL contains an apparently active CoA ligase
(CAL) and ACP domain, and a KR and DH domain with important amino acid
substitutions (Additional file 1:
Figure S1). The putative PKS gene gonP8
consists of a loading module containing a CAL and an ACP domain, and an
extension module with a KS, ATp, KR, DH and ACP domain, all of them with no
significant active site amino acid replacements (Additional file
1: Figure S1).

The proposed biosynthesis pathway of the PM100117/18 NQ moiety
is depicted in Fig. 5. GonM1 (MqnA),
GonM2 (MqnC), GonM4 (MqnD) and a futalosine hydrolase (MqnB) enzyme coded by
a gene external to the PM100117/18 cluster could catalyze the biosynthesis
of DH6N, which would be then methylated by the putative methyltransferase
GonMT to form 3-methyl-DH6N (Fig. 5). Interestingly, GonMT is only 11 % identical to SCO4556,
which is the enzyme proposed to catalyze the quinone-ring C2-methylation in
the last step of MK biosynthesis [33–35].
There is no available information to deduce the possible mechanism by which
3-methyl-DH6N is elongated with a propionate unit to form the NQ moiety.
However, based on the fatty acid activation process catalyzed by fatty
acyl-AMP ligases [39], we could
speculate that the putative synthetase-ligase GonSL could catalyze the
synthesis of a 3-methyl-DH6N-AMP adduct, and the subsequent transfer of
3-methyl-DH6N to the pantetheine group of its own ACP domain. In a next
step, 3-methyl-DH6N might be transferred to the LD of PKS GonP8, and then
elongated by the GonP8 extension module. Binding of 3-methyl-DH6N-AMP to the
GonSL ACP domain, and its following transfer to the LD of PKS GonP8, may
require the participation of any of the putative 3-oxoacyl-ACP synthase III
(KSIII) enzymes coded by genes gonS1 and
gonS2. The involvement of KSIII
proteins in the priming of starter units alternative to malonyl-CoA and
methylmalonyl-CoA has been previously reported in the biosynthesis of a
number of compounds [40–42].
Likewise, the mechanism by which NQ is eventually transferred to PM100117/18
may also require intervention of KSIII, GonP8 or both.

Pathway regulation

Four ORFs (gonMR, gonL1, gonL2 and
gonL3) could be responsible for PM100117/18 pathway
regulation as they code for proteins with high sequence resemblance to
transcription regulatory proteins. GonMR contains a helix-turn-helix (HTH)
motif (smart00347) highly conserved among members of the MarR family of
protein regulators. GonL1 and GonL3 contain a N-terminal nucleoside
triphosphate binding motif (pfam13191) and a HTH domain of the LuxR-type at
de C terminus (smart00421), which are distinctive functional features of the
LAL family of transcription factors [43, 44]. In
addition to a LuxR-like C-terminal HTH motif, GonL2 contains a PAS-sensor
binding fold at the N terminus (cd00130). This domain is designated PAS
because of its homology to the Drosophila
period protein (Per), the aryl hydrocarbon receptor nuclear translocator
protein (ARNT) and the Drosophila
single-minded protein (Sim) [45].

PM100117 and PM100118 transport

ORFs gonT1 and gonT2 code for a putative ATP-binding cassette
(ABC) transport system. ABC transport complexes are comprised of a
hydrophilic protein containing an ATP-binding domain and a hydrophobic
protein with six membrane-spanning domains [46]. GonT1 shares high sequence identity and similarity
with ABC-type membrane permeases and its analysis with the TMHMM server
predicts the formation in this protein of six hydrophobic transmembrane
domains. Conversely, GonT2 displays an ABC-like nucleoside triphosphate
hydrolase domain (cl21455), which in combination with the putative permease
GonT1 could produce a complex to facilitate PM100117/18 transport across the
membrane.

Cluster boundaries analysis

On the cluster left side, genes gonMR,
gonL1, gonL2 and gonL3 were presumed to code for pathway-specific
regulators for PM100117/18 biosynthesis, and orf9, which codes for a putative dioxygenase, was a candidate
gene to accomplish polyketide post-PKS oxygenations. Thus, to verify this
boundary, we performed ultra-performance liquid chromatography (UPLC) analysis
of PM100117/18 production in mutant strains ΔgonL1, ΔgonMR and Δ5201 in
which gonL1, gonMR and orf9, respectively,
have been deleted. The result of this analysis showed that in Δ5201 PM100117/18
production was not altered with respect to S.
caniferus GUA-06-05-006A wild type. By contrast, in ΔgonL1 and ΔgonMR, PM100117/18 biosynthesis was absent or severely decreased
(Fig. 6a). Curiously, deletion of
these genes induced the production of a new compound (NR), with a maximum
absorption wavelength at 260 nm, apparently not related to PM100117/18
biosynthesis. Since PM100117/18 derivatives lacking small functional groups
could possess retention times close to those of the parental products, Δ5201
extracts were also analyzed by LC-MS, confirming that peaks labeled as 1 and 2 indeed
correspond to PM100117 and PM100118, respectively. These results verified the
involvement of genes gonL1 and gonMR in PM100117/18 biosynthesis, presumably
coding for positive pathway-specific transcriptional regulators. PM100117/18
production was partially recovered in mutant strains ΔgonL1 and ΔgonMR when a copy
of gonL1 and gonMR, respectively, was re-introduced (Additional file
1: Figure S2). The cluster left
border is thus apparently defined by gonMR.
Upstream to this gene, antiSMASH detected nine ORFs coding for proteins with
putative functions (Table 1) likely not
pertaining to PM100117/18 biosynthesis. Most of these putative activities are
related to oxidation-reduction reactions, such as ferredoxin (orf7), short-chain dehydrogenase (orf5), 3-ketoacyl-ACP reductase (orf4) and aspartate dehydrogenase (orf1), as well as a cupin domain-containing protein
(orf3).

On the cluster right side, gene gonCP has been demonstrated by genetic engineering to be
involved in the polyketide post-PKS oxygenation. Detailed data on this finding
are described in section below. Adjacent to gonCP, antiSMASH detected ORFs coding for proteins with putative
activities 3-oxoacyl-ACP reductase (orf10),
HxlR family transcriptional regulator (orf11), hypothetical membrane protein (orf12) and NADPH-dependent quinone reductase (orf13). Hence, in order to verify the cluster right
boundary, strains Δ5257, Δ5259 and Δ5261, which lack orf10, orf11 and orf13, respectively, were assessed for PM100117/18
production by UPLC. As shown in Fig. 6b,
deletion of these genes does not have any effect on PM100117/18 biosynthesis,
confirming that they do not belong to the cluster. Therefore, the right cluster
boundary seems to be defined by gene gonCP.

Generation and characterization of novel PM100117/18 derivatives

The main goal of this work was engineering novel PM100117/18
analogues with improved antitumor properties. We first sought to obtain
structural analogues lacking the NQ moiety. For that purpose, a series of mutant
strains affected in NQ biosynthesis putative genes (Fig. 5) was generated by disruption of gonP8, or individual deletion of gonM4, gonMT,
gonSL, gonS1 or gonS2. The resulting
strains, gonP8−, ΔgonM4,
ΔgonMT, ΔgonSL, ΔgonS1 and ΔgonS2, were examined for PM100117/18 production by
UPLC at 254 nm (Fig. 7a). These analyses
did not detect PM100117/18 biosynthesis in gonP8−, ΔgonMT,
ΔgonSL, ΔgonS1 and ΔgonS2. Only in
ΔgonM4 some traces of PM100117/18 were
detected (Fig. 7a). To assess whether
the reason of these changes in the PM100117/18 production level was the absence
or decrease of NQ biosynthesis, we examined the accumulation of PM100117/18
intermediates lacking the NQ moiety in the mutant strains. It is important to
note that loss of the NQ unit causes a change in the maximum absorption
wavelength with respect to the parental compounds, shifting from 254 to 216 nm.
UPLC and LC-MS analysis at 216 nm detected in the six mutant strains several
compounds (Fig. 7b, triangles) with the
expected absorption spectra. In addition, two of these products (3 and 4) possessed
molecular weights compatible with PM100117 [1,
UPLC Rt = 5.182 min, m/z 1601.9 (M + Na)+] and PM1001118 [2, UPLC Rt = 5.536 min, m/z
1585.9 (M + Na)+]
biosynthetic intermediates lacking the NQ moiety. The chemical structures of
compounds 3 [UPLC
Rt = 4.14 min, m/z 1231.7 (M + Na)+] and 4 [UPLC Rt = 4.21 min, m/z 1215.7
(M + Na)+]
were determined by NMR (Additional file 2: Figure S5), confirming that both products correspond to
PM100118 analogues lacking the NQ moiety (Fig. 7c). Interestingly, compound 3 carries an additional hydroxyl group at the aglycone C18,
which is not present in any of the parental products, suggesting that this
derivative could belong to a PM100118 shunt biosynthetic pathway. Mutant
complementation with the corresponding genes partially restored PM100117/18
production (Additional file 1: Figure
S2). These results confirm the involvement of genes gonM4, gonMT, gonSL, gonS1,
gonS2 and gonP8 in the biosynthesis and/or transfer of the NQ unit to
PM100117/18.

Fig. 7

Characterization of genes involved in the biosynthesis
of the PM100117 and PM100118 naphthoquinone unit. Analysis of
PM100117 (1) and PM100118
(2) production by UPLC at
254 nm (a) and 216 nm
(b) in Streptomyces caniferus
GUA-06-05-006A wild type (GUA) and ΔgonM4, ΔgonMT, ΔgonSL,
ΔgonS1, ΔgonS2, and gonP8− mutant strains. Peaks with an
absorption spectrum compatible with PM100117 and PM100118
derivatives lacking the NQ moiety are tagged with triangles. c Chemical structures of PM100118 derivatives
lacking the naphthoquinone moiety. Asterisks indicate the point where the PM100118
chemical structures have been modified

The in vitro antitumor activity of 3 and 4 was determined by
measuring their GI50 (50 % inhibition on cell growth),
TGI (total growth inhibition) and LC50 (50 % cell death)
concentrations [47], against cancer
cell lines A549 (human lung carcinoma cells), PSN1 (pancreas carcinoma),
MDA-MB-231 (human breast adenocarcinoma) and HT29 (human colorectal carcinoma).
The values of these three antitumor indicators for compounds 3 and 4 were
remarkably higher than those of PM100117/18, indicating a weaker cytotoxic
activity of the derivative compounds in comparison with the natural drugs
(Table 2).

Table 2

In vitro antitumor activity of compounds 1–6

Compounds

A549 (µM)

HT29 (µM)

MDA-MB-231 (µM)

PSN1 (µM)

1

PM100117

GI50

1.52

3.04

2.66

nd

TGI

1.84

3.23

2.79

nd

LC50

2.22

3.61

2.97

nd

2

PM100118

GI50

2.24

1.92

1.73

nd

TGI

3.13

2.81

2.75

nd

LC50

4.28

4.09

4.16

nd

3

from mutant gonP8−

GI50

>8.40

>8.40

>8.40

>8.40

TGI

>8.40

>8.40

>8.40

>8.40

LC50

>8.40

>8.40

>8.40

>8.40

4

from mutant gonP8−

GI50

>8.40

>8.40

>8.40

>8.40

TGI

>8.40

>8.40

>8.40

>8.40

LC50

>8.40

>8.40

>8.40

>8.40

5

from mutant ∆gonCP

GI50

0.38

0.13

0.71

0.90

TGI

0.54

1.80

0.83

0.96

LC50

0.77

2.50

1.03

1.03

6

from mutant ∆gonCP

GI50

1.11

3.39

2.60

2.80

TGI

1.17

4.30

2.80

3.00

LC50

1.24

5.48

3.00

3.19

GI50 compound concentration that produces 50 % inhibition on
cell growth as compared to control cells, TGI compound concentration that produces total
growth inhibition as compared to control cells, LC50 compound concentration that produces 50 % cell death as
compared to control cells, Nd
values not determined

Given the cytotoxicity decay caused by the loss of the NQ moiety,
we sought to perform a genetic manipulation leading to minor modifications of
the PM100117/18 structure while preserving the NQ unit. With this aim, we
deleted gonCP, which codes for a putative
cytochrome P450 monooxygenase potentially involved in the oxygenation of the
aglycone C16 and C17 (Fig. 3). UPLC
analysis revealed the ability of the resulting mutant strain, ΔgonCP, to produce several compounds (triangles,
Fig. 8a) with absorption spectra
related to PM100117/18. Furthermore, analysis of fermentation extracts from
ΔgonCP by LC-MS showed that two of these
compounds, 5 [UPLC
Rt = 5.14 min, m/z 1587.9 (M + Na)+] and 6 [UPLC Rt = 5.50 min, m/z 1571.9
(M + Na)+],
possess molecular weights corresponding to PM100117 (1) and PM100118 (2) analogues,
respectively, lacking a keto group. Compounds 5
and 6 were then purified and analyzed by NMR to
determine their chemical structures (Additional file 2: Figure S5), confirming that they derive from PM100117 and
PM100118, respectively, by loss of the C16 aglycone keto functional group
(Fig. 8b). This confirms the
implication of gene gonCP in C16 oxygenation,
but the question on the enzyme that catalyzes C17 hydroxylation still remains.
The production of the natural compounds was restored when gonCP was re-introduced in the ΔgonCP mutant (Additional file 1: Figure S2).

Fig. 8

Characterization of cytochrome P450 monooxygenase gene
gonCP. a UPLC analysis of PM100117
(1) and PM100118
(2) production in
Streptomyces caniferus
GUA-06-05-006A wild type (GUA) and ΔgonCP mutant. Peaks with an absorption spectrum
related to PM100117 and PM100118 derivatives are tagged
with triangles. b Chemical structures of PM100117 and
PM100118 derivatives lacking a keto functional group of the
macrolactone moiety. Asterisks indicate the point where the PM100117
and PM100118 chemical structures have been modified

Likewise compounds 3 and 4, the antitumor activity of derivatives 5 and 6 was examined
against various cancer cell lines. Interestingly, compound 5 possesses an in vitro cytotoxicity threefold to fourfold
higher than its corresponding parental product (1, PM100117) against A549 and MDA-MB-231 cell lines, as
indicated by the lower GI50, TGI and
LC50 concentration values (Table 2). The GI50 concentration
of this compound also showed an outstanding 23-fold increase of antitumor
activity, relative to PM100117, against HT29 tumor cells. However, this result
is not concomitant with a similar decrease of the TGI and
LC50 concentrations, which keep values close to that
of compound PM100117. Additional analysis of 5
by antibiotic activity assays against Micrococcus
luteus and Saccharomyces
cerevisiae further confirms an enhanced bioactivity of this
derivative with respect to the parental products (Additional file 1: Figure S3). PM100118 analogue 6 showed a twofold to threefold improvement in the in
vitro cytotoxic activity against the A549 cell line, as revealed by its lower
GI50, TGI and LC50 values
relative to the natural compound (2,
PM100118).

In this work, we have identified the PM100117 and PM100118 biosynthetic
gene cluster, which has been characterized on the basis of bioinformatics analysis
and genetic engineering data. The cluster spans a region of 169 kb and contains 41
genes coding for all the putative functions presumably required for PM100117/18
biosynthesis (Fig. 2; Table 1). From these activities, biosynthesis of
PM100117/18 can be predicted as follows. Firstly, a type I multimodular PKS
containing a loading module and 20 extension modules, catalyzes the 21 condensation
reactions required for the biosynthesis of the aglycone polyketide chain
(Fig. 3). The macrolatone moiety is then
decorated with two 2,6-dideoxysugar moieties. Next, the NQ unit is transferred to
the second deoxysugar and then glycosylated with the third 2,6-dideoxysugar. The
presence in the cluster of four putative glycosyltransferase genes is consistent
with the PM100117/18 glycosylation pattern. One of the four glycosyltransferases
might transfer l-axenose to the first position
of the glycosylation profile. Two glycosyltransferases might be responsible for the
transfer of l-2-deoxy-fucose (PM100117) or
l-rhodinose (PM100118) to the second
glycosylation position. Finally, the last glycosyltransferase would transfer
l-rhodinose to the NQ moiety. Carbons 16
and 17 of the aglycone contain a keto and a hydroxyl group, respectively. These
oxygenations could be introduced at any step of PM100117/18 biosynthesis, after
completion of the polyketide biosynthesis, as a tailoring modification. We have
shown that the putative cytochrome P450 monooxygenase GonCP is responsible, at
least, for C16 oxygenation (Fig. 8). There
is not a clear candidate for C17 oxygenation, being the closest orf9. Deletion of orf9, which codes for a putative dioxygenase and lies outside the
PM100117/100118 cluster boundaries, does not show any effect on C17 hydroxylation
(Fig. 6). However, the involvement of
gonCP acting as a multifunctional oxygenase
in C17 modification cannot be excluded. The oxygenation of consecutive carbons at
polyketide aglyca by multifunctional cytochrome P450 monooxygenases has been
previously reported [48]. Taking this
into consideration we cannot discard the possibility of orf9 product, or any other oxygenase coding gene at S. caniferus GUA-06-05-006A chromosome, complementing
the putative GonCP C17 oxygenation in ΔgonCP
mutant strain. Four genes code for putative transcriptional regulators belonging to
the MarR (GonMR) and LuxR (GonL1, GonL2 and GonL3) family, indicating that
PM100117/18 biosynthesis might be subjected to a tight transcriptional regulation.
Genetic engineering data suggest that proteins GonMR and GonL1 act as putative
transcriptional activators. This is an interesting finding because MarR
transcriptional activators have been rarely described in the literature
[49]. However, other mechanisms can
also be considered regarding the mode of action of GonMR, such as a dual role as
activator-repressor [50] or
co-activator.

The noticeable structural similarity of the NQ unit to MK induced us to
envisage a connection between the biosynthesis of both compounds (Fig. 5). This hypothesis is further supported by the
presence in the cluster of four genes (gonM1,
gonM2, gonM3 and gonM4), coding for
proteins highly identical to those involved in the early steps of MK biosynthesis
via futalosine [33–35]. Based on their assigned putative functions,
GonM1 (MqnA), GonM2 (MqnC) and GonM4 (MqnD) could catalyze three of the four
reaction steps leading to the biosynthesis of DH6N, which, according to our model,
defines the branching point towards NQ and MK biosynthesis (Fig. 5). Even though C2-methylation of the NQ quinone ring
could occurs at any step of its biosynthesis, it is very plausible that this
reaction delivers DH6N to the NQ branch. Deletion of gonM4 diminishes PM100117/18 production, and leads to the
accumulation of biosynthetic intermediates lacking the NQ unit (Fig. 7c). This result confirms the involvement of
gonM4 in NQ biosynthesis. However, in
contrast to gonP8−, ΔgonMT, ΔgonSL, ΔgonS1 and
ΔgonS2 mutant strains, in which PM100117/18
biosynthesis is completely abolished, in ΔgonM4 a
low PM100117/18 production level can be detected (Fig. 7a). This indicates that the additional mqnD homologue, present outside the PM100117/18 gene cluster, is
functional and able to partially complement the loss of gonM4. We do not fully understand the role of duplicated MqnA, MqnC
and MqnD gene functions in the genome. However, we can speculate that the presence
in the cluster of dedicated mqnA, mqnC and mqnD genes
leading to DH6N biosynthesis could offers two main advantages. One is to provide
sufficient supply of DH6N, which is probably a limiting intermediate as it is common
to MK and NQ biosynthesis. The second advantage could be to facilitate a coordinated
regulation with other genes involved in PM100117/18 biosynthesis, thus ensuring an
optimal PM100117/18 production when these are required. To date, this is the first
time that MK biosynthesis is described as a nexus between secondary and primary
metabolism. Nonetheless, the existence of duplicated gene homologues in a single
genome, one being part of the primary metabolism and the other present in a
secondary metabolites gene cluster, has been frequently described for genes
(ccr) coding for crotonyl-coA
carboxylase/reductase. Multiple ccr homologous
have been identified in gene clusters for the biosynthesis of various polyketide
natural products [51–54]. As we have speculated for the presence of
MK biosynthesis genes in the PM100117/18 gene cluster, the role of ccr duplications has also been postulated to be the
supply of sufficient precursor building blocks for polyketide biosynthesis
[51].

The generation of PM100117/18 structural analogues with improved
antitumor activity relative to their natural products, represents a substantial
achievement of this work. These derivatives were accomplished by a genetic
manipulation, gonCP deletion, leading to a minor
modification of PM100117/18 structure, the loss of a keto functional group. The
twofold to fourfold improvement of antitumor activity showed by these derivatives is
an interesting finding because the generation of truncated products, or even the
removal of minor structural elements of a natural compound, often affects
bioactivity negatively [55]. For
instance, deletion of a gene coding for a cytochrome P450 monooxygenase of the
pimaricin gene cluster in S. natalensis conduces
to the production of analogue 4,5-deepoxypimaricin, which differs from the natural
compound in a single oxygenation and shows a diminished antibacterial activity
relative to the parental product [56].
Similar results have been reported on the pikromycin analogues neopikromycin and
narbomycin produced by S. venezuelae, which lack
a single hydroxyl group at different positions of the polyene ring and possess a
noticeably reduced antibacterial activity [57]. Instead, most of the reported genetic engineering
approaches, leading to the generation of improved bioactive analogues, consist of
the addition or replacement of structural components in the parental product
[17, 58]. In this regard, cytochrome P450 monooxygenases have been
frequently contemplated as promising targets for engineering the biosynthesis of
novel therapeutic natural compounds. As an example, replacement of the C16 carboxyl
on the nystatin analogue S44HP with a methyl group by mutation of a P450
monooxygenase gene, yielded a twofold more active antifungal analogue [59]. Herein, compounds 5 and 6 has been only assessed for
in vitro cytotoxicity against different tumor cell lines. However, aside from an
enhanced bioactivity, structural modifications frequently yield derivatives with
other additional desirable pharmacological properties, such as lower toxicity or
improved solubility [59–61]. In future works some of these properties of
compounds 5 and 6
might also be addressed.

On the other hand, the loss of the NQ unit caused in vitro cytotoxicity
decay, indicating that this moiety is central to PM100117/18 antitumor activity.
Curiously, two compounds structurally related to PM100117/18, langkolide
[26] and GT35 [27], which harbor a similar napthtoquinone
moiety, also possess cytotoxic activity. Furthermore, the anti-proliferative effect
of MKs on tumor cells both in vitro and in vivo has been repeatedly reported
[62, 63]. Conversely, other macrolides structurally resembling
PM100117/18 but lacking a napthtoquinone unit, such as liposidolide A [64] and polaramycin [65], exhibit antifungal and antibacterial but
not antitumor activity. All together, these observations suggest the idea that
PM100117/18 cytotoxic activity could, to large extent, stems from the NQ
moiety.

The availability of the PM100117 and PM100118 gene cluster, and the
genetic insights into their biosynthesis, will help to understand how similar
natural compounds are produced. This information enables the engineering of more
derivatives with improved pharmacological properties such as an enhanced biological
activity.

PM100117 and PM100118 are members of a group of glycosylated compounds
hallmarked by the presence in their structures of a NQ chromophore structurally
resembling MK. Our results show that biosynthesis of the NQ chromophore is a complex
process that involves diverse enzymes and which is connected to primary metabolism.
The presence in secondary metabolite gene clusters of certain primary metabolism
genes may be explained in terms of a sufficient supply of limiting intermediates.
This connection of secondary metabolism with MK biosynthesis has never been reported
before. A similar situation might be encountered in the future when other gene
clusters for natural products structurally related to PM100117/18 will be
characterized. On the other hand, the analysis of PM100117/18 analogues has shown
interesting insights into the structure-bioactivity relationship of these family of
natural products. Removal of the C16 keto group leads to an increased antitumor
activity of both the PM100117 and PM100118 derivative. However, the overall
cytotoxicity level showed by compound 5 is higher
than the observed in compound 6. This suggests that
PM100117 could be a more promising target to undertake other structural
modifications. In addition, based on the results presented in this work and
previously described data on similar compounds, the presence of NQ moieties might be
a predictive structural feature of cytotoxic activity. This issue could be taken
into account at future screening for novel antitumor natural products.

Analysis of metabolite production and compound purification

Samples (3 ml) from S. caniferus
GUA-06-05-006A whole-cultures (see above) were mixed with an equal volume of
ethyl acetate and incubated at room temperature for 2 h. The organic phase was
then recovered by centrifugation (3000×g,
10 min) and evaporated in vacuo. The residue
was dissolved in methanol:DMSO (1:1) to perform UPLC and LC-MS analyses as
described elsewhere [68].

For the purification of compounds 3, 4, 5 and 6, mycelia of the
corresponding producing strains were separated from the culture by
centrifugation and extracted twice with ethyl acetate. The supernatants were
filtered and applied to a solid-phase extraction cartridge (Sep-Pak Vac C18,
10 g, Waters) that had been fitted with a perforated stopper pierced by a
stainless steel HPLC tubing. The culture broth was applied by means of a
peristaltic pump and subsequently the cartridge was connected to a HPLC
quaternary pump (model 600E, Waters). The retained material was eluted with a
mixture of methanol and 0.05 % trifluoroacetic acid (TFA) in water. A linear
gradient from 0 to 100 % methanol in 60 min, at 10 ml/min, was used. Fractions
were taken every 5 min, collected on 5 ml of 0.1 M phosphate buffer, pH 7.0 and
analyzed by UPLC. Those fractions containing the desired compounds were
evaporated in vacuo and subsequently
re-dissolved in a small volume of a mixture of DMSO and methanol (50:50). The
organic extract of the culture pellets was similarly evaporated and
re-dissolved. The compounds of interest were purified by preparative HPLC using
a SunFire C18 column (10 µm, 10 × 250 mm, Waters). Compounds were
chromatographed with mixtures of acetonitrile or methanol and 0.05 % TFA in
water in isocratic conditions optimized for each peak, at 7 ml/min, and were
always collected on 0.1 M phosphate buffer, pH 7.0. Compound 5 was purified with 55 % acetonitrile in a first step
and with 82 % methanol in a second step. Compound 6 was purified with 55 % acetonitrile in a first step and with
85 % methanol in a second step. Compounds 3 and
4 were purified with 32 % acetonitrile in a
first step and with 37 % acetonitrile in a second step. After every purification
step, the collected compounds were diluted fourfold with water and then applied
to a solid-phase extraction cartridge (Sep-Pak C18, Waters). The cartridge was
washed with water, the retained compound was eluted with methanol and dried
in vacuo. Once the purification was
finished, the compounds were dissolved in a mixture of tert-butanol and water
(1:1) and lyophilized.

In vitro cytotoxicity assay

Triplicate cultures were incubated for 72 h in the presence or
absence of test compounds (at ten concentrations ranging from 10 to
0.0026 mg/mL). For quantitative estimation of cytotoxicity, the colorimetric
sulforhodamine B (SRB) method was used [69]. Briefly, cells were washed twice with PBS, fixed for
15 min in 1 % glutaraldehyde solution, rinsed twice in PBS, and stained in 0.4 %
SRB solution for 30 min at room temperature. Cells were then rinsed several
times with 1 % acetic acid solution and air-dried. Sulforhodamine B was then
extracted in 10 mM trizma base solution and the absorbance measured at 490 nm.
Using the mean ± SD of triplicate cultures, a dose-response curve was
automatically generated using nonlinear regression analysis. Three reference
parameters were calculated (NCI algorithm) by automatic interpolation:
GI50 = compound concentration that produces 50 % cell
growth inhibition, as compared to control cultures; TGI = total cell growth
inhibition (cytostatic effect), as compared to control cultures, and
LC50 = compound concentration that produces 50 % net
cell killing (cytotoxic effect).

Mass spectra and structural elucidation

(+)-HRESIMS was performed on an Agilent 6230 time of flight LC/MS.
NMR spectra were recorded on a Varian “Unity 500” spectrometer at 500/125 MHz
(1H/13C). Chemical
shifts were reported in ppm using residual CD3OD (d 3.31
for 1H and 49.0 for 13C)
as internal reference. HMBC experiments were optimized for a
3JCH of 8 Hz. ROESY spectra were measured with a mixing
time of 500 ms. The structures were established by
1H- and 13C-NMR and two
dimensional NMR experiments correlation spectroscopy (COSY), heteronuclear
multiple quantum coherence (HMQC), heteronuclear multiple-bond correlation
(HMBC).

DNA manipulation and plasmids construction

The isolation and manipulation of DNA were carried out following
standard general methods previously described for E.
coli [70] and
Sreptomyces [66]. PCR amplifications were conducted by
using Herculase II Fusion polymerase (Agilent Technologies) with a touchdown PCR
procedure. The termocycler (SureCycler 8800, Agilent Technologies) was
programmed as follow: initial denaturation at 99.9 °C for 4 min; 20 cycles of
99.9 °C for 20 s, 65–45 °C touchdown for 20 s and 72 °C for tx (20 s/kb) min
followed by 10 cycles of 99.9 °C for 20 s, 60 °C for 20 s and 72 °C for tx
(20 s/kb) min. Final extension was performed at 72 °C for 3 min. PCR products of
the expected size were gel-purified and sequenced.

A detailed description of the construction of plasmids used in this
work can be found in Additional file 3: Methods S1. Plasmids for inactivation of genes gonP1 and gonP8
were constructed in the conjugative plasmid pOJ260 [71], which lacks the capacity to replicate
in Streptomyces and carries the aac(3)IV gene marker that confers resistance to apramycin
(ApmR). To achieve the single deletion of genes
gonM4, gonMT, gonSL, gonS1, gonS2,
gonCP, gonMR, gonL1, orf9, orf10,
orf11 and orf13, DNA sequences flanking the target genes were amplified
with the primer pairs designated in Additional file 3: Table S1 and cloned at both sides of the aac(3)IV gene in plasmid pEFBA-oriT [72]. The hygromycin B resistance
(HygR) gene marker, hyg, was then extracted from plasmid pLHyg [73] and introduced in the deletion plasmids
(Additional file 3: Methods S1). Gene
hyg allows recognizing clones in which a
complete gene replacement by a double cross-over has taken place
(HygS ApmR) from those
in which a single cross-over event has integrated the deletion plasmid into the
chromosome (HygR ApmR). A
suitable plasmid backbone to accomplish the complementation of
ApmR mutants was constructed as follows. The
integrative plasmid pSETec [68],
which harbors the constitutive ermE*p
promoter, was digested with NcoI. A 1.6 Kb fragment containing hyg was extracted from pLHyg by NheI/SpeI
digestion. The linearized pSETec plasmid and the pLHyg NheI-SpeI fragment were
then blunt-ended with the Klenow fragment of DNA polymerase I and ligated to
afford plasmid pSETHe. Complementation plasmids were generated by inserting the
target genes into the XbaI/EcoRV sites of pSETHe, under the transcriptional
control of the ermE*p promoter (Additional
file 3: Methods S1).

Sequencing and bioinformatics analysis

The S. caniferus GUA-06-05-006A
chromosome was sequenced at Lifesequencing Ltd., Valencia, Spain by Roche/454
pyrosequencing [74] on a Genome
Sequencing FLX platform. The genome was assembled in the Newbler assembler
package [75] version 2.8 by using
default parameters. Identification of gene clusters for biosynthesis of
secondary metabolites was performed by the antibiotics and secondary metabolite
analysis shell: antiSMASH 3.0.4 [22]. Annotation of ORFs within the PM100117/18 biosynthesis gene
cluster was based on database searching of the corresponding proteins carried
out by BLAST algorithm [76] at the
National Center for Biotechnology Information (NCBI). Additional sequence
alignments were conducted by ClustalW2 [77] and EMBOSS needle [78] from the European Molecular Biology Laboratory (EMBL).
Prediction of transmembrane domains was performed by TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/).

The nucleotide sequence of the PM100117/18 gene cluster was
deposited in GeneBank (accession numbers: LN997801 and LN997802).

Authors’ contributions

JAS, FDC, CM and CO conceived and designed the project; RGS and CG conducted
experiments and analyzed the data; RGS and CO performed sequence in silico analysis; AFB carried out compound
purifications; RF performed structural elucidation of novel derivatives; RGS drafted
the manuscript and CO, JAS and CM contributed to preparing the final version of the
paper. All authors read and approved the final manuscript.

Acknowledgements

This work was supported by the Spanish Ministry of Economy and
Competitiveness in the INNPACTO programme 2012–2015, with the project number
IPT-2011-0752-900000 (Bioketido).

Competing interests

The authors declare that they have no competing interests.

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